Effect of Nitric Oxide Donors on Anthrax Lethal Toxin Inactivation in Rat Model

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Anthrax infection remains a bioterrorist health threat today capable of affecting a large number of individuals in a relatively brief period. Defining effective but inexpensive therapies that can be stock-piled for prolonged periods and quickly administered on a large scale would be of great value for the US public in the event of an anthrax outbreak. Production of anthrax lethal toxin (LeTx) is central to the pathogenesis of anthrax. This is a binary type toxin consisting of an A (lethal factor, LF) and B component (protective antigen, PA). PA is necessary for cellular uptake of the toxic moiety LF. Lethal factor is a zinc dependent protease, which inactivates all but one mitogen activated protein kinase kinase (MPKK 1 to 4 and 6). These MPKKs participate in signaling pathways necessary for cell survival. We and other labs have shown that LeTx can produce shock and lethality similar to that observed during live bacterial infection. Importantly, inhibition of the cellular uptake of LeTx with selective antibodies to PA is protective in bacteria challenged anthrax models, emphasizing the important contribution this toxin makes to the lethality of anthrax. Despite their demonstrated effectiveness however, antibody preparations are expensive and may be difficult to administer on a large scale. Furthermore, bioengineered strains of anthrax capable of producing toxin insensitive to the effects of such antibody preparations can be manufactured with relatively few resources. Developing alternative agents capable of inhibiting anthrax toxin is warranted. In prior in vivo work we found that LeTx was a potent inhibitor of endogenous nitric oxide (NO) production in lethal lipopolysaccharide (LPS) and E. coli challenged rats. While such inhibition during anthrax infection itself may promote bacterial growth by suppressing host innate immune responses, it may aid the microbe in other ways as well. Consistent with this, in in vivo experiments in the rat lab, we observed that nonlethal doses of LPS capable of rapidly increasing NO levels were actually protective with lethal LeTx challenge. These in vivo findings suggest that administration of NO donor agents may be of therapeutic benefit during anthrax infection. Depending on type, NO donor agents can be produced inexpensively, stored in large quantities, and can be orally bioavailable. Such agents could be administered relatively efficiently on a large scale during an anthrax outbreak. The present protocol is designed to investigate the potential of NO donor agents for the treatment of LeTx associated shock. As a proof of principle investigation, it has focused on agents that can be administered intravenously to ensure delivery. The study has consisted of two parts thus far. In the first part (Study 1), the ability of three different NO donors sodium nitroprusside (SNP), S-nitroso-L-glutathione (GSNO) and 3-morpholinosdnonime (SIN-1) to produce increases in circulating NO levels that are measurable (>15% of control) and sustainable over 24 hours with <15% decreases in mean arterial blood pressure (MBP) will be tested in a catheterized rat model. This data was employed to develop a regimen of SIN-1 treatment directed against LeTx. The second part of the protocol (Study 2), has tested the ability of SIN-1 treatment to inhibit the lethal effects of LT. In initial experiments we showed that pretreatment of LT with SIN-1 did inhibit LTs in vivo lethal effects. Subsequent experiments showed that coinfusion of SIN-1 with LT (i.e. simultaneously via the same venous catheter) was significantly protective, presumably inactivating LT during the process of infusion. Two final sets of experiments have now been completed in which: 1) SIN-1 and LT have been infused via separate catheters in LT challenged animal and 2) animals were treated with a NOS inhibitor and then challenged with LT. Data from these experiments is under analysis anda manuscript is in preparation. Data from the study was presented at the International Conference of theAmerican Thoracic Society 2012 and 2014. A manuscript is in preparation.